Amyotrophic lateral sclerosis (ALS) is characterized by the progressive and selective loss of motor neurons (MNs) in the motor cortex, brainstem, and spinal cord, resulting in death of patients typically 2-5 years after diagnosis. Glutamate-mediated excitotoxicity in the CNS has been implicated as a major contributor to MN cell death in ALS. It is thought that the vulnerability of MNs to glutamate-mediated excitotoxicity is due to a combination of their high density of Ca2+ permeable ion channels, and their diminished capacity to handle excessive Ca2+ influx, which serves as a trigger for neurodegeneration. Why MNs of ALS patients are especially vulnerable to excitotoxicity remains unknown, but there is nearly universal acceptance that excitotoxicity contributes to selective death on MNs in ALS patients. Therefore, the development of drugs which lower Ca2+ influx during excitatory activity in the CNS, is a crucial focal point in the pursuit for therapeutics for ALS. Ionotropic AMPA receptors are glutamate-activated ion channels that are key mediators of Ca2+ influx in MNs. AMPA receptors are composed of four subunits termed GluR1-4. Two alternatively spliced variants of all four GluRs called flip and flop are normally expressed in the CNS and are known to modulate AMPA channel conductance. AMPA channels containing GluR-flip isoforms are 'higher gain' channels, which have greater current amplitudes and/or greater resistant to glutamate-desensitization than the 'lower gain' AMPA channels containing flop variants. Thus, when the flip/flop ratios of GluRs are elevated, AMPA receptors in MNs show enhanced excitatory activity, and greater Ca2+ influx. We recently developed several novel chemically-modified RNA oligonucleotides, called splice modulating oligomers (SMOs), which potently and specifically modulate GluR alternative splicing to reduce the flip/flop ratio of various GluR isoforms. Although SMOs do not cross the blood-brain-barrier, after direct delivery into the CSF they are broadly distributed throughout the CNS and have the extraordinary capacity to readily to cross the membrane and enter the nuclei of cells in the CNS, where their splice modulating activity persists for months. Here we propose a translational set of experiments to validate the potential of our SMOs as therapeutics for treating ALS. First, we will compare the efficacy of our lead SMO (LSP-GR3) in reducing GluR3-flip expression throughout the CNS, using (1) continuous ICV delivery, and (2) single bolus injection into the lumbar sac of the spinal column (Aim 1). We will then use the best delivery modality determined in Aim 1 and evaluate the efficacy of our two lead SMOs to improve motor function, and increase lifespan in ALS mice (Aim 2).